Accelerated nucleic acid reassociation method

Claim 1

A method for the formation of a double-stranded nucleic acid molecule from separate single-stranded nucleic acid molecules wherein the rate of formation is increased at least 100 times over the rate of formation of a double-stranded nucleic acid molecule from the same single-stranded nucleic acid molecules in a solution using 0.18M NaCl at 60° C. at the same pH, said method comprising the steps of: preparing an aqueous reaction solution containing, in solution, a first quantity of a first single-stranded nucleic acid molecule and a second quantity of a second single-stranded nucleic acid molecule, said second single-stranded nucleic acid molecule having base sequences complementary to sequences of said first single-stranded nucleic acid molecule so as to allow for the formation of the double-stranded molecule, and a concentration of at least one nucleic acid precipitating agent dissolved therein consisting essentially of an amount sufficient to precipitate said first and second single-stranded nucleic acid molecules so as to increase said rate of formation at least 100 times the formation rate for the same double-stranded nucleic acid molecule in said reaction solution, comprising 0.18M NaCl in said reaction solution in place of said agent, at 60° C.; and incubating said aqueous reaction solution at a temperature at which reassociation can occur.

Claim 2

A method for the formation of a double-stranded nucleic acid molecule from separate single-stranded nucleic acid molecules wherein the rate of formation is increased at least 100 times over the rate of formation of a double-stranded nucleic acid molecule from the same single-stranded nucleic acid molecules in a solution using 0.18M NaCl at 60° C. at the same pH, said method comprising the steps of: preparing an aqueous reaction solution containing, in solution, a first quantity of a first single-stranded nucleic acid molecule and a second quantity of a second single-stranded nucleic acid molecule, said second single-stranded nucleic acid molecule having base sequences complementary to sequences of said first single-stranded nucleic acid molecule so as to allow for the formation of the double-stranded molecule, and a concentration of at least one nucleic acid precipitating agent dissolved therein consisting essentially of an amount sufficient to precipitate said first and second single-stranded nucleic acid molecules so as to increase said rate of formation at least 100 times the formation rate for the same double-stranded nucleic acid molecule in said reaction solution, comprising 0.18M NaCl in said reaction solution in place of said agent, at 60° C.; and incubating said aqueous reaction solution at a temperature at which reassociation can occur.

Claim 3

A method for the formation of a double-stranded nucleic acid molecule from separate single-stranded nucleic acid molecules wherein the rate of formation is increased at least 100 times over the rate of a formation of a double-stranded nucleic acid molecule from the same single-stranded nucleic acid molecules in solution using 0.18M NaCl at 60° C. at the same pH, said method comprising the steps of: preparing an aqueous reaction solution containing, in solution, a first quantity of a first single-stranded nucleic acid molecule and a second quantity of a second single-stranded nucleic acid molecule, said second single-stranded nucleic acid molecule having base sequences complementary to base sequences of said first single-stranded nucleic acid molecule so as to allow for the formation of the double-stranded molecule mixing said aqueous reaction solution with a second solution having at least one nucleic acid precipitating agent, such that the resulting mixture has a concentration of said nucleic acid precipitating agent dissolved therein consisting essentially of an amount sufficient to precipitate said first and second single-stranded nucleic acid molecules so as to increase the rate of formation at least 100 times the formation rate for the same double-stranded nucleic acid molecule in said reaction solution comprising 0.18M NaCl in said reaction solution in place of said agent, at 60° C. and; incubating the resulting mixture at a temperature at which reassociation can occur.

Claim 4

A method for the formation of a double-stranded nucleic acid molecule from separate single-stranded nucleic acid molecules wherein the rate of formation is increased at least 100 times over the rate of a formation of a double-stranded nucleic acid molecule from the same single-stranded nucleic acid molecules in solution using 0.18M NaCl at 60° C. at the same pH, said method comprising the steps of: preparing an aqueous reaction solution containing, in solution, a first quantity of a first single-stranded nucleic acid molecule and a second quantity of a second single-stranded nucleic acid molecule, said second single-stranded nucleic acid molecule having base sequences complementary to base sequences of said first single-stranded nucleic acid molecule so as to allow for the formation of the double-stranded molecule mixing said aqueous reaction solution with a second solution having at least one nucleic acid precipitating agent, such that the resulting mixture has a concentration of said nucleic acid precipitating agent dissolved therein consisting essentially of an amount sufficient to precipitate said first and second single-stranded nucleic acid molecules so as to increase the rate of formation at least 100 times the formation rate for the same double-stranded nucleic acid molecule in said reaction solution comprising 0.18M NaCl in said reaction solution in place of said agent, at 60° C. and; incubating the resulting mixture at a temperature at which reassociation can occur.

Claim 5

A method for forming a double-stranded nucleic acid molecule from a first and second single-stranded nucleic acid molecule; wherein said first and second nucleic acid molecules comprise first and second nucleic acid sequences respectively which are sufficiently complementary to cause said first and second nucleic acid molecules to hybridize at a first rate, to form the double-stranded nucleic acid molecule, in an aqueous solution comprising 0.18M NaCl and no other salt at 60° C., comprising the steps of: (a) providing an aqueous solution comprising said first and second nucleic acid molecules, and a concentration of a nucleic acid precipitating agent in an amount sufficient to precipitate said first and second nucleic acid molecules at 60° C. and thereby cause said first and second nucleic acid molecule to hybridize at a second rate, said second rate being at least 100 times said first rate, and (b) incubating said aqueous solution at a temperature at which said first and second nucleic acid molecules hybridize at a rate at least 100 times said first rate.

Claim 6

A method for forming a double-stranded nucleic acid molecule from a first and second single-stranded nucleic acid molecule; wherein said first and second nucleic acid molecules comprise first and second nucleic acid sequences respectively which are sufficiently complementary to cause said first and second nucleic acid molecules to hybridize at a first rate, to form the double-stranded nucleic acid molecule, in an aqueous solution comprising 0.18M NaCl and no other salt at 60° C., comprising the steps of: (a) providing an aqueous solution comprising said first and second nucleic acid molecules, and a concentration of a nucleic acid precipitating agent in an amount sufficient to precipitate said first and second nucleic acid molecules at 60° C. and thereby cause said first and second nucleic acid molecule to hybridize at a second rate, said second rate being at least 100 times said first rate, and (b) incubating said aqueous solution at a temperature at which said first and second nucleic acid molecules hybridize at a rate at least 100 times said first rate.

Claim 29

A method for detecting the presence of a bacterium in a sample suspected of containing said bacterium, said method comprising: preparing an aqueous reaction solution containing, in solution, a first quantity of a test sample suspected of containing said bacterium, said test sample having been treated with a quantity of a denaturing agent sufficient to disassociate the double-stranded nucleic acid molecules present in said bacterium into single-stranded nucleic acid molecules, a second quantity of a second single-stranded nucleic acid molecule complementary to the base sequence of a nucleic acid molecule of said bacterium to be detected so as to allow for the formation of a double-stranded molecule, and a concentration of at least one nucleic acid precipitating agent dissolved therein consisting essentially of an amount sufficient to precipitate said first and second single-stranded nucleic acid molecules so as to increase the rate of formation at least 100 times the formation rate for the same said double-stranded nucleic acid molecule in said reaction solution, comprising 0.18M NaCl in said reaction solution in place of said agent, at 60° C.; incubating said aqueous reaction solution at a temperature at which reassociation can occur; and, assaying said incubated aqueous reaction solution for the presence or amount of said double-stranded nucleic acid molecule.

Claim 30

A method for detecting the presence of a bacterium in a sample suspected of containing said bacterium, said method comprising: preparing an aqueous reaction solution containing, in solution, a first quantity of a test sample suspected of containing said bacterium, said test sample having been treated with a quantity of a denaturing agent sufficient to disassociate the double-stranded nucleic acid molecules present in said bacterium into single-stranded nucleic acid molecules, a second quantity of a second single-stranded nucleic acid molecule complementary to the base sequence of a nucleic acid molecule of said bacterium to be detected so as to allow for the formation of a double-stranded molecule, and a concentration of at least one nucleic acid precipitating agent dissolved therein consisting essentially of an amount sufficient to precipitate said first and second single-stranded nucleic acid molecules so as to increase the rate of formation at least 100 times the formation rate for the same said double-stranded nucleic acid molecule in said reaction solution, comprising 0.18M NaCl in said reaction solution in place of said agent, at 60° C.; incubating said aqueous reaction solution at a temperature at which reassociation can occur; and, assaying said incubated aqueous reaction solution for the presence or amount of said double-stranded nucleic acid molecule.

Claim 44

A method as defined in any one of claims 1, 2, 19, and 26 wherein the nucleic acid precipitating agent concentration is sufficient so as to increase the rate at least 1000 times the rate in 0.18M NaCl at 60° C.